Project Summary Rotaviruses (RVs) are the most common cause of severe gastroenteritis and dehydrating diarrhea in in- fants and young children worldwide. Despite the wide administration of several vaccines, RVs still remain a highly significant human pathogen, leading to over 215,000 deaths annually. The inadequate understanding of RV-host interaction greatly impedes the development of improved vaccines and therapeutic interventions. In the preliminary studies, Dr. Ding has performed an unbiased genome-wide CRISPR-Cas9 screen and a high-throughput RV-host proteomic interactome mapping, to bridge this gap in knowledge and identified stromal antigen 2 (STAG2) as a novel host factor required for RV infection. The infectivity of human and animal RVs was significantly reduced in STAG2-/- cells. STAG2 is an integral member of the nuclear cohesin complex and how it promotes cytoplasmic RV replication is perplexing and intriguing. In this application, using a set of powerful and tractable model systems, Dr. Ding will test the hypothesis that RV hijacks the STAG2/cohesin complex to block DNA damage and inhibit IFN induction, thereby enhancing virus infection. During the K99 phase, Dr. Ding will scrutinize the signaling pathways in STAG2-/- cells that mediate resistance to RV infection (Aim 1; K99). Dr. Ding will further generate a new mouse model that specifically lacks Stag2 in the intestinal epithelium (Aim 2; K99) and employ the novel human intestinal organoids (Aim 2; R00) to study how STAG2 deficiency affects RV infection in a physiologically valid environment. Finally, Dr. Ding will follow up on his pro- vocative finding that several subunits of the cohesin complex physically bind to an RV non-structural protein. He will determine the outcome of such interaction for RV replication and pathogenesis in vivo, using a new re- verse genetics system (Aim 3; R00). Collectively, these studies on RV-cohesin interaction will constitute the scientific basis for the rational design of antiviral inhibitors (by transiently targeting STAG2) and vaccine candi- dates (RV mutants incapable of cohesin interaction), which will be further pursued in future R01 applications. Dr. Ding has developed a comprehensive career development plan, under the direct mentorship of Dr. Harry Greenberg at Stanford University, to perfect his training in experimental (human organoids) and profes- sional (lab management and grantsmanship) skills to achieve his short-term goals of successfully carrying out the proposed project and establishing independence. A prestigious advisory committee (Drs. Ann Arvin, Peter Jackson and Jan Carette at Stanford), in combination with a team of excellent external consultants and collab- orators, will provide essential guidance in genomic screens, proteomics, and mouse genetics, to complement Dr. Ding's strong background in virology and immunology. The exemplary environment in the Department of Microbiology and Immunology at the Stanford School of Medicine, will enable Dr. Ding to fully benefit from this Award and attain his long-term career goal to lead a world-class laboratory studying enteric viruses with a spe- cial focus on the viral pathogenesis and interaction with the host intestinal innate immune system.